Clinically actionable neoantigens in non-small cell lung cancer Lung cancer is the leading cause of cancer death among US Veterans as well as the world's leading cause of cancer death. Environmental exposure and tobacco use among Veterans operate together to increase risk. Unleashing the immune response against pulmonary premalignancy could transform therapy and outcomes. Here, we propose to lay the ground work for lung ?cancer interception,? a strategy that seeks to block the progression of premalignancy to invasive cancer. In our preliminary studies we have begun to evaluate the mutational landscape of pulmonary premalignancy and the associated premalignant microenvironment by whole exome DNA sequencing and immunohistochemistry. Remarkably, we find frequent immune-effector cell infiltration as well as evidence of immune suppression in pulmonary premalignancy. These findings lead us to hypothesize that premalignant-associated neoantigens (PANs) are recognized and elicit immune responses at the earliest points of lung adenocarcinoma development. This research will identify neoepitopes that can be targeted before the development of invasive lung cancer, thus shifting the approach to disease interception through immunoprevention and treatment of the very earliest phase of the disease. The specific aims are: 1) To utilize whole exome DNA sequencing (WES) to determine computationally-defined neoantigens in matched sets of primary tumor, premalignant lesions and adjacent histologically normal lung tissues. 2) To identify functionally relevant neoepitopes associated with tumor progression: Two sources of T cells, one from TIL, the other from peripheral blood PD1+ T cells, will be used to identify neoepitope-specific CD8+ T cells. To improve screening efficiency, we will focus on neoantigens shared between pre-malignant lesions and primary tumors that are potentially progression-relevant. Functional assays will be performed to verify the identified neoepitopes. This will lead to patient-tailored neoepitopes for future vaccine or adoptive cell therapies for non-small cell lung cancer (NSCLC). 3) To relate the immune contexture to WES-defined mutational landscapes in premalignancy and the associated tumor we will: 3A) Perform quantitative multiplexed immunofluorescent staining of the same specimens utilized in the first two aims to assess the regulators of cell- mediated immunity and to relate this to the mutational landscapes and neoepitopes of the premalignant lesions and tumors, 3B) Evaluate gene expression utilizing a Nanostring panel of 770 immune-related genes and, 3C) Integrate findings from WES, gene expression and tissue immunostaining to define the landscape of adenocarcinoma pulmonary premalignancy. This research seeks to transform the approach to both the prevention and treatment of lung adenocarcinoma by discovery of functional neoepitopes that can be utilized in the development of vaccines and adoptive therapies to intercept disease at the earliest phases. With the advent of screening, many patients are presenting with imaging studies consistent with focal or multifocal premalignancy. As we increase our knowledge of the molecular pathogenesis and natural history of the disease, discovery of critical neoepitopes will facilitate vaccine development for those at the highest risk for progression to invasive lung cancer.